WO2022260772A1 - Lipid nanoparticle formulations for gastrointestinal delivery - Google Patents

Abstract

Lipid-containing particles and drug products containing the lipid-containing particles are provided. Methods of local and systemic delivery of therapeutic agents to, and through, mucosa, such as intestinal mucosa, are provided.

Description

LIPID NANOPARTICLE FORMULATIONS FOR GASTROINTESTINAL DELIVERY

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of United States Provisional Patent Application No. 63/208,588 filed June 9, 2021 , the disclosure of which is incorporated herein by reference in its entirety.

[0002] Lipid nanoparticles are efficient carriers of cargo, such as a nucleic acid cargo, for delivery into cells for gene delivery, mRNA delivery, antisense, RNA interference, among other uses. Lipid nanoparticles typically comprise helper lipids, cholesterol, ionizable lipids (e.g., lipidoids), lipid-polymer conjugates and nucleic acid cargo. Lipid nanoparticles are typically administered in an intravenous, intramuscular or subcutaneous injection. Exemplary LNP compositions and/or compositions, e.g., lipidoids, useful in producing LNPs are described in U.S. Patent Nos. 10,844,028, 10,189,802, 9,872,911 , 9,556,110, 9,439,968, 9,227,917, 8,969,353, and 8,450,298, as well as in U.S. Patent Application Publication Nos. 2017/0204075, 2019/0177289, 2017/0152213, 2016/0114042, 2015/0203439, 2014/0322309, 2014/0161830,

2011/0293703, and 2010/0331234, each of which is incorporated herein by reference for its technical disclosure relating to compounds and compositions useful in delivery of nucleic acid cargoes, and to the extent it is consistent with the present disclosure. Additional examples of lipid nanoparticles are described in U.S. Patent Nos. 9,404,127, 9,364,435, and US 8,058,069, each of which incorporated herein by reference for its technical disclosure relating to compounds and compositions useful in delivery of nucleic acid cargoes, and to the extent it is consistent with the present disclosure (see, also, e.g., Sabnis S, et al., A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. Mol Ther. 2018;26(6):1509-1519 and Yonezawa S, et al., Recent advances in si RNA delivery mediated by lipid-based nanoparticles. Adv Drug Deliv Rev. 2020;154-155:64-78). The BNT162b2 and mRNA-1273 COVID9 vaccines are formulated as lipid nanoparticles. Examples of lipid nanoparticles, lipidoids, and methods of making lipid nanoparticles and lipidoids, as described herein, are described in Whitehead KA, et al., Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat Commun. 2014 Jun 27; 5:4277. doi: 10.1038/ncomms5277. PMID: 24969323; PMCID: PMC4111939. [0003] Despite some degree of success in parenteral delivery of nucleic acids via LNPs, there are significant obstacles to delivery of nucleic acids to mucosa, e.g., in the gastrointestinal tract, including significant pH changes, and the need to penetrate mucus.

SUMMARY

[0004] It was found that certain lipid nanoparticle formulations penetrate mucosa. As such, methods of trans-mucosal delivery of cargoes, such as nucleic acids, are provided.

[0005] A trans-mucosal drug delivery method is provided for delivery of a therapeutic agent to a patient. The method comprises administering to mucosa of a patient a composition comprising a lipid-containing particle, such as a lipid nanoparticle, comprising a therapeutic agent, the lipid-containing particle comprising: a helper lipid having a negative charge at pH7; cholesterol or a derivative thereof; a PEGylated fatty acid-containing compound , such as a PEG-containing polymeror a PEGylated phospholipid, such as C - phosphatidylethanolamine; and an ionizable lipidoid, e.g., that forms a cation at an acidic pH; wherein the lipid-containing particle has a negative surface charge at pH 7, and optionally pH 5.

[0006] An oral drug product is provided. The drug product comprises: a core comprising a lipid-containing particle, such as a lipid nanoparticle, comprising a therapeutic agent, the lipid-containing particle comprising: a helper lipid having a negative charge at pH7; cholesterol; a PEG-based compound, such as a PEG-containing polymer or a PEGylated phospholipid, such as C -phosphatidylethanolamine (commercially-available, e.g., from Avanti Polar Lipids); and an ionizable lipidoid, e.g., that forms a cation at an acidic pH; wherein the lipid-containing particle has a negative surface charge at pH7, and optionally pH 5; and an enteric or timed-release coating covering the core. BRIEF DESCRIPTION OF THE DRAWINGS [0007] FIGS. 1A and 1B. FIG. 1A provides a general reaction scheme between an amine, e.g., the numbered compounds shown in FIGS. 2A-2C, and an acrylate tail, e.g., the compounds designated 0_, as shown in FIGS. 3A - 3C to form a lipidoid, which is referenced by the reacted amine and the reacted acrylate tail (###0_), e.g., 3060io, as shown in FIG. 1B. The compounds may be prepared by the addition of a primary or secondary amine to an acrylate via a Michael addition reaction.

[0008] FIGS. 2A-2C provide exemplary amines for use in preparing lipidoids as described herein.

[0009] FIGS. 3A-3C provide exemplary acrylates for use in preparing lipidoids as described herein.

[0010] FIG. 4 is a table depicting lipidoid compositions, e.g., as tested in Example 1 , with the amine moiety referenced by number (see, FIGS. 2A-2C) on the left, and the acrylate (See FIGS. 3A-3C) listed across the top.

[0011] FIG. 5 is a schematic diagram of an exemplary dosage form.

[0012] FIG. 6 provides schematic diagrams of the transfection efficacy assay in Caco- 2 intestinal epithelial cells with and without a mucin layer.

[0013] FIG.7: LNP transfection of Caco-2 intestinal epithelial cells covered with mucin. [0014] FIGS. 8A and 8B provide graphs showing that the best Caco-2-transfecting LNPs have low TNS at pH7 and pH5, respectively.

[0015] FIGS. 9A and 9B provide graphs showing that the best mucin-coated Caco-2- transfecting LNPs have low TNS at pH7 and pH5, respectively.

[0016] FIG. 10 provides graphs showing TNS fluorescence for samples with 16 mol% helper lipid and 40% helper lipid at pH 7 and 5. Helper lipid charge affects TNS fluorescence, indicating changes in overall LNP charge.

[0017] FIG. 11 shows gas propulsion chemistries enhance transit of LNPs across a mucin covered transwell.

[0018] FIG. 12 shows lipid nanoparticles efficiently transfect gastrointestinal tract following intestinal injection.

[0019] FIG. 13 shows lipid nanoparticles efficiently transfect liver, spleen and pancreas following intestinal injection.

[0020] FIG. 14 is a graph showing that lipid nanoparticles consistently transfect intestines following intestinal injection. [0021] FIG. 15 is a graph showing that lipid nanoparticles consistently transfect liver following intestinal injection.

[0022] FIG. 16 is a graph showing that lipid nanoparticles consistently transfect spleen following intestinal injection.

[0023] FIG. 17 is a graph showing that lipid nanoparticles consistently transfect pancreas following intestinal injection.

[0024] FIG. 18 provides a graph showing the effect of different helper lipids and polymers on the efficacy of an exemplary formulation as provided herein.

[0025] FIG. 19 provides a graph showing the effect of different helper lipids and polymers on the efficacy of an exemplary formulation as provided herein.

[0026] FIG. 20 provides a graph showing the effect of different buffers/antacids on the efficacy of an exemplary formulation as provided herein.

[0027] FIG. 21 provides a graph showing the effect of different buffers/antacids on the efficacy of an exemplary formulation as provided herein.

[0028] FIG. 22 is a graph showing the effect of Cio fatty acid as a partial substitute for cholesterol on the efficacy of an exemplary formulation as provided herein.

[0029] FIG. 23 is a graph showing the effect of Cio fatty acid as a partial substitute for cholesterol on the efficacy of an exemplary formulation as provided herein.

[0030] FIG. 24 is a graph showing the effect of bile acids as a substitute for cholesterol on the efficacy of an exemplary formulation as provided herein.

[0031] FIG. 25 is a graph showing the effect of bile acids as a substitute for cholesterol on the efficacy of an exemplary formulation as provided herein.

[0032] FIG. 26 is a graph showing the effect of buffer/antacid on the efficacy of an exemplary formulation as provided herein in an oral gavage experiment.

DETAILED DESCRIPTION

[0033] Other than in the operating examples, or where otherwise indicated, the use of numerical values in the various ranges specified in this application are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.

[0034] As used herein, “a” and “an” refer to one or more. [0035] The term “comprising” is open-ended and may be synonymous with “including”, “containing”, or “characterized by”. The term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting of” excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments “comprising” one or more stated elements or steps also include, but are not limited to embodiments “consisting essentially of” and “consisting of” those stated elements or steps. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases.

[0036] As used herein, the terms “patient” or “subject” refer to members of the animal kingdom including but not limited to human beings and “mammal” refers to all mammals, including, but not limited to human beings.

[0037] "Treatment" in the context of a disease or disorder, a marker for a disease or a disorder, or a symptom of a disease or disorder, can refer to a clinically-relevant and/or a statistically significant decrease or increase in an ascertained value for a clinically- relevant marker from outside a normal range towards, or to, a normal range. The decrease or increase can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, to a level accepted as either a therapeutic goal, or a level within the range of normal for an individual without such disease or disorder, or, in the case of a lowering of a value, to below the level of detection of an assay. The decrease or increase can be to a level accepted as within the range of normal for an individual without such disease or disorder, which can also be referred to as a normalization of a level. The reduction or increase can be the normalization of the level of a sign or symptom of a disease or disorder, that is, a reduction in the difference between the subject level of a sign of the disease or disorder and the normal level of the sign for the disease or disorder (e.g., to the upper level of normal when the value for the subject must be decreased to reach a normal value, and to the lower level of normal when the value for the subject must be increased to reach a normal level).

[0038] . The compositions described herein may include as an active agent, a nucleic acid reagent, such as, without limitation, a DNA, an RNA (e.g., an mRNA), an antisense reagent or an RNAi (RNA interference) reagent.

[0039] As used herein, the terms “cell” and “cells” refer to any types of cells from any animal, such as, without limitation, rat, mouse, monkey, and human. For example and without limitation, cells can be progenitor cells, e.g., pluripotent cells, including stem cells, induced pluripotent stem cells, multipotent cells, or differentiated cells, such as endothelial cells and smooth muscle cells. “Cells” may be in vivo, e.g., as part of a tissue or organ, or in vitro, such as a population of cells, such as, for example, a population of cells enriched for a specific cell type, such as, without limitation, a progenitor cell or a stem cell.

[0040] A composition is “biocompatible” in that the composition and, where applicable, elements thereof, or degradation products thereof, are substantially non-toxic to cells or organisms within acceptable tolerances, including substantially non-carcinogenic and substantially non-immunogenic, and are cleared or otherwise degraded in a biological system, such as an organism (patient) without substantial toxic effect. Non limiting examples of degradation mechanisms within a biological system include chemical reactions, hydrolysis reactions, and enzymatic cleavage.

[0041] "Therapeutically effective amount," as used herein, can include the amount of an lipid-containing particle, such as an LNP, as described herein that, when administered to a subject having a disease, can be sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the lipid-containing particle, such as an LNP, how the composition is administered, the ultrasound treatment protocol, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0042] A "therapeutically-effective amount" can also include an amount of an agent that produces a local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. Lipid-containing particle, such as an LNP, employed in the methods described herein may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

[0043] The phrase "pharmaceutically-acceptable carrier" as used herein can refer to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier can be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1 ) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21 ) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (24) other non-toxic compatible substances employed in pharmaceutical formulations.

[0044] A “group” or “functional group” is a portion of a larger molecule comprising or consisting of a grouping of atoms and/or bonds that confer a chemical or physical quality to a molecule. A “residue” is the portion of a compound or monomer that remains in a larger molecule, such as a polymer chain, after incorporation of that compound or monomer into the larger molecule. A “moiety” is a portion of a molecule, and can comprise one or more functional groups, and in the case of an “active moiety” can be a characteristic portion of a molecule or compound that imparts activity, such as pharmacological or physiological activity, to a molecule as contrasted to inactive portions of a molecule such as esters of active moieties, or salts of active agents. [0045] As used herein, the term “polymer composition” is a composition comprising one or more polymers. As a class, “polymers” includes, without limitation, homopolymers, heteropolymers, copolymers, block polymers, block co-polymers and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, whereas copolymers contain more than one type of monomer.

[0046] A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer that the polymer comprises is not the same as the monomer prior to incorporation into the polymer, in that at the very least, during incorporation of the monomer, certain groups, e.g., terminal groups, that are modified during polymerization are changed, removed, and/or relocated, and certain bonds may be added, removed, and/or modified. An incorporated monomer is referred to as a “residue” of that monomer. A polymer is said to comprise a specific type of linkage if that linkage is present in the polymer. Unless otherwise specified, molecular weight for polymer compositions refers to weight average molecular weight {Mw). A “moiety” is a portion of a molecule, compound or composition, and includes a residue or group of residues within a larger polymer. [0047] As used herein, "alkyl" refers to straight, branched chain, or cyclic hydrocarbon groups including, for example, from 1 to about 20 carbon atoms, for example and without limitation C1-3, C1-6, C1-10 groups, for example and without limitation, straight, branched chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like. An alkyl group can be, for example, a Ci, C2, C3, C4, C5, C6, C7, Cs, C9, C10, C11 , C12, C13, CM, C15, C16, C17, C18, C19, C20, C21 , C22, C23, C24, C25, C26, C27, C28, C29, C30, C31 , C32, C33, C34, C35, C36, C37, C38, C39, C40, C41 , C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted “lower alkyl” refers to C1-C6 alkyl. Non-limiting examples of straight alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Branched alkyl groups comprises any straight alkyl group substituted with any number of alkyl groups. Non-limiting examples of branched alkyl groups include isopropyl, n-butyl, isobutyl, sec-butyl, and f-butyl. Non-limiting examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptlyl, and cyclooctyl groups. Cyclic alkyl groups also comprise fused-, bridged-, and spiro- bicycles and higher fused-, bridged-, and spiro-systems. A cyclic alkyl group can be substituted with any number of straight, branched, or cyclic alkyl groups. “Unsaturated alkyl” may comprise one or more, e.g., 1 , 2, 3, 4, or 5, carbon-to-carbon double bonds and alternatively may be referred to as alkene or alkenyl, as described below. "Substituted alkyl" can include alkyl substituted at 1 or more (e.g., 1 , 2, 3, 4, 5, 6, or more) positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein. "Optionally substituted alkyl" refers to alkyl or substituted alkyl. "Halogen," "halide," and "halo" refers to -F, -Cl, -Br, and/or -I. "Alkylene" and "substituted alkylene" can include divalent alkyl and divalent substituted alkyl, respectively, including, without limitation, methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, hepamethylene, octamethylene, nonamethylene, or decamethylene. "Optionally substituted alkylene" can include alkylene or substituted alkylene.

[0048] "Alkene or alkenyl" can include straight, branched chain, or cyclic hydrocarbyl groups including, e.g., from 2 to about 20 carbon atoms, such as, without limitation Ce-24 groups in the case of fatty acids, having one or more, e.g., 1 , 2, 3, 4, or 5, carbon- to-carbon double bonds, and may be referred to as “unsaturated alkyl” in the context of fatty acids an lipids. The olefin or olefins of an alkenyl group can be, for example, E, Z, cis, trans, terminal, or exo-methylene. An alkenyl or alkenylene group can be, for example, a C2, C3, C4, Cs, C6, C7, Cs, C9, C10, C11 , C12, C13, CM, C15, C16, C17, Cis, C19, C20, C21 , C22, C23, C24, C25, C26, C27, C28, C29, C30, C31 , C32, C33, C34, C35, C36, C37, C38, C39, C40, C41 , C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. A halo-alkenyl group can be any alkenyl group substituted with any number of halogen atoms. "Substituted alkene" can include alkene substituted at 1 or more, e.g., 1 , 2, 3, 4, or 5 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein. "Optionally substituted alkene" can include alkene or substituted alkene. Likewise, "alkenylene" can refer to divalent alkene. Examples of alkenylene include without limitation, ethenylene (-CH=CH-) and all stereoisomeric and conformational isomeric forms thereof. "Substituted alkenylene" can refer to divalent substituted alkene. "Optionally substituted alkenylene" can refer to alkenylene or substituted alkenylene.

[0049] An "ester" is represented by the formula -OC(0)R, where R can be an alkyl, alkenyl, or group described above.

[0050] Alkyne or "alkynyl" refers to a straight, branched chain, or cyclic unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. The triple bond of an alkyne or alkynyl group can be internal or terminal. Examples of a (C2-C8)alkynyl group include, but are not limited to, acetylene, propyne, 1 -butyne, 2- butyne, 1 - pentyne, 2-pentyne, 1 -hexyne, 2-hexyne, 3-hexyne, 1 -heptyne, 2-heptyne, 3-heptyne, 1 -octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below. An alkyne or alkynyl group can be, for example, a C2, C3, C4, Cs, Ce, C7, Cs, C9, C10, C11, C12, C13, CM, C15, C16, C17, Cis, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, C47, C48, C49, or C50 group that is substituted or unsubstituted. A halo-alkynyl group can be any alkynyl group substituted with any number of halogen atoms. The term "alkynylene" refers to divalent alkyne. Examples of alkynylene include without limitation, ethynylene, propynylene. "Substituted alkynylene" refers to divalent substituted alkyne.

[0051] “PEG” refers to polyethylene glycol. “PEGylated” refers to a compound comprising a moiety, comprising two or more consecutive ethylene glycol moieties. Non-limiting examples of PEG moieties for PEGylation of a compound include, one or more blocks of from 1 to 200 ethylene glycol units, such as -(0-CH2-CH2)n-, -(CH2- CH2-0)n-, or -(0-CH2-CH )n-0H, where n ranges, for example and without limitation, from 1 to 200 or from 1 to 100, for example from 1 to 5, or 1 .

[0052] “Aryl," alone or in combination refers to an aromatic ring system such as phenyl or naphthyl. "Aryl" also can include aromatic ring systems that are optionally fused with a cycloalkyl ring. A "substituted aryl" is an aryl that is independently substituted with one or more substituents attached at any available atom to produce a stable compound, wherein the substituents are as described herein. The substituents can be, for example, hydrocarbyl groups, alkyl groups, alkoxy groups, and halogen atoms. "Optionally substituted aryl" refers to aryl or substituted aryl. An aryloxy group can be, for example, an oxygen atom substituted with any aryl group, such as phenoxy. An arylalkoxy group can be, for example, an oxygen atom substituted with any aralkyl group, such as benzyloxy. "Arylene" denotes divalent aryl, and "substituted arylene" refers to divalent substituted aryl. "Optionally substituted arylene" refers to arylene or substituted arylene. A “polycyclic aryl group” and related terms, such as “polycyclic aromatic group” refers to a group composed of at least two fused aromatic rings. “Heteroaryl” or “hetero-substituted aryl” refers to an aryl group substituted with one or more heteroatoms, such as N, O, P, and/or S. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0053] “Cycloalkyl" refers to monocyclic, bicyclic, tricyclic, or polycyclic, 3- to 14-membered ring systems, which are either saturated, or partially unsaturated. The cycloalkyl group may be attached via any atom. Cycloalkyl also contemplates fused rings wherein the cycloalkyl is fused to an aryl or hetroaryl ring. Representative examples of cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below. “Cycloalkylene" refers to divalent cycloalkyl. The term "optionally substituted cycloalkylene" refers to cycloalkylene that is substituted with at least 1 , 2 or 3 substituents, attached at any available atom to produce a stable compound, wherein the substituents are as described herein.

[0054] “Carboxyl” or “carboxylic” refers to group having an indicated number of carbon atoms, where indicated, and terminating in a -C(0)0H group, thus having the structure -R-C(0)0H, where R is an unsubstituted or substituted divalent organic group that can include linear, branched, or cyclic hydrocarbons. Non-limiting examples of these include: Ci-s carboxylic groups, such as ethanoic, propanoic, 2- methylpropanoic, butanoic, 2,2-dimethylpropanoic, pentanoic, etc. “Amine” or “amino” refers to group having the indicated number of carbon atoms, where indicated, and terminating in a -NH2 group, thus having the structure -R-NH2, where R is a unsubstituted or substituted divalent organic group that, e.g., includes linear, branched, or cyclic hydrocarbons, and optionally comprises one or more heteroatoms. The term “alkylamino” refers to a radical of the formula -NHR^X or -NR^XR^X where each R^x is, independently, an alkyl radical as defined above.

[0055] Terms combining the foregoing refer to any suitable combination of the foregoing, such as arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylhereroaryl. As an example, “arylalkylene" refers to a divalent alkylene wherein one or more hydrogen atoms in an alkylene group is replaced by an aryl group, such as a (C3-Cs)aryl group. Examples of (C3-C8)aryl-(Ci-C6)alkylene groups include without limitation 1 -phenylbutylene, phenyl-2-butylene, l-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, and naphthylethylene. The term "(C3-C8)cycloalkyl-(Ci-C6)alkylene" refers to a divalent alkylene wherein one or more hydrogen atoms in the C1-C6 alkylene group is replaced by a (C3-C8)cycloalkyl group. Examples of (C3-C8)cycloalkyl-(Ci-C6)alkylene groups include without limitation 1 -cycloproylbutylene, cycloproyl-2-butylene, cyclopentyl-1 -phenyl-2-methylpropylene, cyclobutylmethylene and cyclohexylpropylene.

[0056] A fatty acid is an aliphatic monocarboxylic acid, comprising a carboxyl group linked to an aliphatic hydrocarbyl group which may be saturated or unsaturated. A hydrocarbyl or hydrocarbon group refers to a group of carbon and hydrogen atoms, such as alkyl, alkenyl (alternatively, unsaturated alkyl), or aryl groups. By “aliphatic”, it is meant acyclic or cyclic, saturated or unsaturated hydrocarbon compounds, excluding aromatic compounds. The aliphatic group of fatty acids is typically a linear chain of carbons, but fatty acids and substituted fatty acids as a class include linear, branched, and/or cyclic carbon chains. As used herein, fatty acids include both natural and synthetic aliphatic carboxylic acids. Fatty acids can have an aliphatic chain of from three to 40 carbon atoms (for example, as used herein, “a (C3-C40) fatty acid”). Hydrogen atoms of a compound, such as a fatty acid may be substituted with a group or moiety (hereinafter referred to as a “substituent”), to produce a substituted fatty acid. Fatty acids and substituted fatty acids may be referred to as “optionally substituted fatty acids”) Fatty acids, and fatty acid groups, may be referred to by the number of carbon atoms and the number of double bonds, e.g., C10:0, referring to a fatty acid or fatty acid group having 10 carbon atoms and zero double bonds. Likewise, C18:1 refers to a fatty acid with an 18-carbon chain having one double bond, such as oleic acid.

[0057] Unsaturated fatty acids and substituted unsaturated fatty acids (collectively “optionally substituted unsaturated fatty acids”) comprise one or more carbon-carbon double bonds, or an alkenyl group (e.g., vinyl group) in their aliphatic chains. The individual carbon atoms of the alkenyl group are referred to herein as alkenyl carbons. Unless specified, any carbon-carbon double bond in the alkyl chain of the described optionally substituted unsaturated fatty acids independently may be E ( trans ) or Z (c/s) geometric isomers, or mixtures thereof.

[0058] Fatty acids may include, without limitation: C3, C4, C5, C6, C7, C8, C9, C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 , C22, C23, C24, C25, C26, C27, C28, C29, C30, C31 , C32, C33, C34, C35, C36, C37, C38, C39, and C40 fatty acids. The fatty acids may be saturated (zero double bonds), or unsaturated, e.g., with 0 or 1 , 2, 3, 4, 5, 6, or more double bonds. Non-limiting examples of saturated fatty acids include: propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid. Non-limiting examples of unsaturated fatty acids include: crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-y-linolenic acid, eicosatrienoic acid, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, cervonic acid, and herring acid.

[0059] Compounds described herein, including fatty acids and substituted fatty acids can exist in various isomeric forms, including configurational, geometric, and conformational isomers, as well as existing in various tautomeric forms, such as those that differ in the point of attachment of a hydrogen atom. The term “isomer” is intended to encompass all isomeric forms of a compound of this invention, including tautomeric forms of the compound.

[0060] Certain compounds described here may have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound can be in the form of an optical isomer or a diastereomer. Accordingly, compounds described herein include their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture unless otherwise specified. Optical isomers of the compounds of the invention can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology, or via chemical separation of stereoisomers through the employment of optically active resolving agents.

[0061] Unless otherwise indicated, “stereoisomer” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.

[0062] Lipids, as a group, include glycerides and phospholipids. A “glyceride” is an ester of glycerol (propane 1 ,2,3-triol) with a fatty acid or a substituted fatty acid. Phospholipids are lipids containing phosphoric acid as mono- or di-esters, such as phosphatidic acids and phosphoglycerides. Phosphoglycerides are di-esters of glycerol, which are glycerol derivatives in which one hydroxyl group of the glycerol is phosphodiester-linked to a group, such as a functional group, such as, for example and without limitation, a 2-amino ethanol or a choline (e.g., -0-CH2-CH2-N⁺(CH3)3) groups. A phosphatidylcholine is a phosphoglyceride with a choline linked to the glycerol moiety by a phosphodiester linkage. A glycerol-phosphoethanolamine is a phosphoglyceride with an 2-amino ethane group (e.g., -CH2-CH2-NH3) linked to the glycerol moiety by a phosphodiester linkage. Amphipathic refers to a molecule or compound having both hydrophobic and hydrophilic parts, e.g., under physiological conditions.

[0063] Lipid particles are provided having an overall negative charge. Examples of lipid-containing particles, as described herein comprise, without limitation: a helper lipid, such as negatively-charged amphipathic phospholipid or phosphoglyceride, such as a phosphatidylserine, a phosphatidylglycerol, a phosphatidic acid, a negatively-charged lyso-lipid, a fatty acid, a bile acid or a conjugated bile acid, an anionic glycolipid, an anionic glycosphingolipid, an anionic glycoglycerophospholipid, or another molecule with a hydrophobic component or tails attached to a net negatively charged moiety or moieties; cholesterol; a PEGylated fatty acid, or a PEGylated phospholipid, such as C - phosphatidylethanolamine (commercially-available, e.g., from Avanti Polar Lipids); and an ionizable lipid, such as an ionizable lipid forming a cation at an acidic pH, e.g., less than 5.

[0064] The lipid-containing particles may be described as lipid nanoparticles or lipid microparticles, depending on their size. The particles may be used to deliver any compatible cargo or active agent, such as, without limitation, a polynucleotide, a drug, a protein or peptide, a small molecule, or a gas. The particles may be used to deliver an anionic or polyanionic cargo to and through mucosa, such as a patient’s gastrointestinal tract. The anionic or polyanionic cargo may be a protein or a peptide. The anionic or polyanionic cargo may be a nucleic acid, such as, without limitation: an mRNA, an antisense reagent, an RNAi agent, a genetic vector or recombinant construct such as a plasmid or other extrachromosomal or chromosome-targeting nucleic acid, a nucleic acid for use in gene editing, a recombinant or natural viral genome, DNA comprising a gene, a ribozyme, or an aptamer. For example and without limitation, the agent or cargo may be an RNA (e.g., mRNA, an RNAi reagent, a dsRNA, an siRNA, an shRNA, an miRNA, an antisense RNA, a guide RNA (gRNA), a long non-coding RNAs (IncRNA), a base editing gRNA (beRNA), a prime editing gRNA (pegRNA), or a transfer RNA (tRNA)). The cargo may be an mRNA, e.g., a capped and optionally PEGylated mRNA, encoding a therapeutic polypeptide or protein, or an immunogen, such as mRNAs encoding a coronavirus spike protein or one or more epitope-containing portions thereof, such as, for example the BNT162b2 and mRNA-1273 mRNAs currently used for immunization against SARS-CoV-2, or future sequence variants thereof.

[0065] The lipid particles described herein may also be incorporated into drug delivery devices, e.g., drug products, dosage forms, unit dosage forms, etc. the lipid particles may be used to encapsulate agents including polynucleotides, small molecules, proteins, peptides, metals, organometallic compounds, etc.

[0066] “Nucleic acids” include DNA and RNA as is found naturally, and chemically- modified nucleic acids, as are broadly-known, but optionally may not contain, as a class, peptide-nucleic acids (PNAs) having a neutral backbone, though modified peptide nucleic acids that are modified with anionic moieties, such as gamma-modified PNAs, may find use in the present compositions and methods. Nucleic acids useful in the compositions and methods described herein may be polyanionic nucleic acids, having an overall negative charge under neutral or physiological conditions, such as in an aqueous solution pH 6-8, e.g., in water, blood, serum, Ringer’s, or normal saline. A nucleic acid may comprises a phosphorus-containing moiety, such as a phosphate and/or a phosphorothioate moiety, and therefore would be polyanionic. Non-limiting examples of nucleic acids include RNAi agents, antisense reagents, aptamers, and ribozymes, among others (see, e.g., Bajan S, Hutvagner G. RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs. Cells. 2020 Jan 7;9(1 ):137; Invitrogen RNAi Handbook, ThermoFisher Scientific 2015; and Kilanowska, A., etal., In vivo and in vitro studies of antisense oligonucleotides - a review, RSC Adv., 2020, 10, 34501 ). Nucleic acids may be unmodified (e.g., natural) or chemically-modified (see, e.g., Dar, S., et at., siRNAmod: A database of experimentally validated chemically modified siRNAs. Sci Rep 6, 20031 (2016) and crdd.osdd.net/servers/sirnamod/).

[0067] Provided herein is a lipid-containing particle, e.g., a lipid nanoparticle or microparticle, formulation for gastrointestinal delivery. Lipid nanoparticles that incorporate negatively-charged helper lipids or other lipid components aid the nanoparticles in traveling through the negatively charged mucus barrier. These negative components could include negatively charged helper lipids such as phosphatidylserine, phosphatidylglycerol, or phosphatidic acid, a negatively charged lyso-lipid, a fatty acid, a bile acid or conjugated bile acid, or another molecule with a hydrophobic component or tails attached to a net negatively charged moiety or moieties.

[0068] Helper lipids may include, for example and without limitation: negatively- charged amphipathic phospholipid or phosphoglyceride, such as a phosphatidylserine, a phosphatidylglycerol, a phosphatidic acid, a negatively-charged lyso-lipid, a fatty acid, a bile acid or a conjugated bile acid, an anionic glycolipid, an anionic glycosphingolipid, or an anionic glycoglycerophospholipid. These compounds, may have one or more fatty acid side chains, which may be the same or different when two or more fatty acid side chains are present, and which may be any fatty acid as described herein. The helper lipid may be a phosphatidylserine. Helper lipids may be combined in LNPs.

[0069] Bile acids are typically found in the bile of mammals and other vertebrates, and include, without limitation: cholic acid, taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, ursodeoxycholic acid, salts of deoxycholic acid and lithocholic acid, and derivatives of any the preceding, among others. Bile acids are commercially available from Avanti Polar Lipids of Birmingham Alabama. Bile acids may be combined in LNPs.

[0070] A net negative charge refers generally to a negative charge, e.g., a negative surface charge, at neutral or physiological pH (e.g., 7.0 or 7.4, or from 7.0 to 7.5). Typical lipid nanoparticle formulations use amphiphilic, net neutral lipids such as phosphatidylethanolamines and phosphatidylcholines. Due to their external positive charge, those amphiphilic or net neutral lipids are prone to binding to the negative charges in mucin and being cleared from the gastrointestinal tract.

[0071] The net charge of the lipid particle may be measured by any useful method or assay. Zeta-potential (z-potential) is one way of measuring surface charge, but may not be sufficiently sensitive in all instances to measure net surface charge of the lipid particles described herein. As such, other methods of determining surface charge may be employed, such as the TNS method. The TNS method uses 2-(p-toluidinyl) naphthalene-6-sulphonic acid (TNS), which only binds to cationic lipids and is commonly used to determine lipid pKa (see, e.g., Uebbing L, et al., Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering. Langmuir. 2020 Nov 10, 36 (44):13331 -13341 ). The TNS value for the lipid-containing particle at pH7 may be less than a TNS value at pH7 for a control lipid-containing particle consisting of:

16% wt. DOPE;

46.5% wt. cholesterol;

2.5% wt. C-I4-PEG2000 phosphatidylethanolamine (PE);

35% wt. 3060iio; and mRNA at a 10:1 lipidoid:mRNA ratio (w/w)% wt.

[0072] The diameter of the lipid-containing particles may range from 1 micrometer to 1 ,000 micrometers (microparticles). The diameter of the particles range may range from 1 micrometer to 100 micrometers, from 1 micrometer to 10 micrometers, from 10 micrometers to 100 micrometers, from 100 micrometer to 1 ,000 micrometers, or from 1 -5 micrometers. The diameter of the lipid particles may range from between 1 nm to 1 ,000 nm (nanoparticles), from 1 nm to 100 nm, from 1 nm to 10 nm, from 10 nm td 00 nm, from 100 nm to 1 ,000 nm, from 20 nm to 2,000 nm, or from 1 to 5 nm. The diameter of the particles range from between 1 pm to 1 ,000 pm, from 1 pm to 100 pm, from 1 pm to 10 pm, from 10 pm to 100 pm, from 100 pm to 1 ,000 pm, or from 1 to 5 pm.

[0073] The lipid particles may be prepared using any useful method. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, and simple and complex coacervation, among other methods. The method of preparing the particles may be the double emulsion process and spray drying. The conditions used in preparing the particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness”, shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may also depend on the agent being encapsulated and/or the composition of the matrix. Methods developed for making particles for delivery of encapsulated agents are amply described in the literature. In one example, the lipid- containing particles are prepared by microfluidics (see, e.g., Chen D, et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012 Apr 25;134(16):6948-51 and Cayabyab, C, et al., “mRNA Lipid Nanoparticles: Robust low-volume production for screening high-value nanoparticle materials,” Document ID: mrnaspark-AN-1018, (2018) Precision NanoSystems, Inc., describing methods of making lipid nanoparticles, including suitable ratios for various constituents). Briefly, appropriate amounts of the lipidoid, the helper lipid, the cholesterol or cholesterol derivative and PEG-based material are mixed in an appropriate solvent, such as 90% ethanol and 10% 10 mM sodium citrate and mixed with an appropriate amount of the cargo, such as siRNA in 10mM sodium citrate at a weight ratio of siRNA or mRNA to the (lipidoid + cholesterol or cholesterol derivative + helper lipid + PEG-based material) of, for example and without limitation of 1 :2-1000, such as from 1 :4 to 1 :50, e.g., 1 :10. For siRNA and mRNA, the lipidoid:siRNA ratio may range from 2:1 to 20:1 , for example for siRNA, the lipidoid:si RNA ratio may be 5:1 , and for mRNA, the lipidoid:mRNA ratio may be 10:1 . The amount of helper lipid in the lipid particle may range from 10 to 80 mol% of the amounts of total lipids, e.g., lipidoid + cholesterol or cholesterol derivative + helper lipid + PEG-based material in the lipid particle. The lipid particles may be formed in a microfluidics device or by rapid pipetting. Particles may be diluted in a suitable aqueous solvent, such as PBS, and optionally dialyzed against the same or a different aqueous solvent. [0074] If the particles prepared by any of the above methods have a size range outside of the desired range, the particles can be sized, for example, using a sieve or filter. The particle may also be coated. The particle may be coated with a targeting agent. [0075] The lipid-containing particles comprise cholesterol or a derivative thereof, such as 3b[N — (N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol). The lipid-containing particles comprise a PEG (poly(oxyethylene))-based material, such as a PEGylated fatty acid-containing compound or PEG-containing block copolymer, such as a polaxamer. Non-limiting examples of PEG-based materials include: PEG- ceramide, PEG-DMG, PEG-PE, poloxamer, or DSPE carboxy PEG. For instance, in certain embodiments, the PEG-based material is C14 PEG2000 DMG, C15 PEG2000 DMG, C16 PEG2000 DMG, C18 PEG2000 DMG, C14 PEG 2000 ceramide, C15 PEG2000 ceramide, C16 PEG2000 ceramide, C18 PEG2000 ceramide, C14 PEG2000 PE, C15 PEG2000 PE, C16 PEG2000 PE, C18 PEG2000 PE, C14 PEG350 PE, C14 PEG5000 PE, poloxamer F-127, poloxamer F-68, poloxamer L-64, or DSPE carboxy PEG.

[0076] A lipidoid is a lipid-like molecule. An ionizable lipidoid is a lipidoid that forms an ion in acidic or basic conditions. Non-limiting examples of ionizable lipidoids are provided in US Patent No. 9,439,968, generally forming lipidoids by conjugate addition of alkyl-acrylates to amines. A general synthesis scheme of useful amino-lipidoids prepared from amines and alkyl-acrylates is shown in FIG. 1A. Also provided are useful amines, e.g., designated as 25, 32, 306, etc., and structures of alkyl-acrylates, e.g., Oio, On, O12, O13, and O . Lipidoids are designated in the examples below in reference to the amine and alkyl-acrylate used to make the ionizable lipidoid, e.g., 3O6O10, referring to A/^S-aminopropylj-A^-methylpropane-l ,3-diamine conjugated to decyl acrylate , as shown in FIG. 1 B, with the technical name for 306010 being tetrakis(decyl) 3,3’,3”,3”’-(((methylazanediyl)bis(propane-3,1 -diyl))bis(azanetriyl)) tetrapropionate.

[0077] Lipidoids for preparation of LNPs for delivery to mucosa may include any combination, e.g., by Michael’s addition, of an alkylamine having from one to five amine moieties, and an alkyl or alkenyl acrylate having a pKa in the range of from 3 to 7. Lipidoids for preparation of LNPs for delivery to mucosa may include any combinatorial permutation of an amine depicted in FIGS. 2A-2C, and one or more acrylate depicted in FIGS. 3A-3C, e.g., as depicted in FIGS. 1 A and 1 B. Lipidoids for preparation of lipid-containing particles for delivery to mucosa may include any combinatorial permutation of an amine depicted in FIGS. 2A-2C, and one or more acrylate depicted in FIGS. 3A-3C, having a pKa in the range of from 3 to 7, for example from 5 to 7, for example and without limitation, as depicted in FIG. 4 (grayed boxes on grid). Additional examples of lipidoids are described in US Patent Application Publication Nos. US20110256175A1 and US20200109113A1 , and US Patent Nos. US7939505B2, US8802863B2, US8969353B2, US9139554B2, and US9227917B2, incorporated herein by reference for their description of additional exemplary lipidoid compounds, and uses therefor.

[0078] Also described herein are methods and formulations, e.g., drug products that enable ionizable lipid nanoparticles to overcome the acid barrier of the gastrointestinal tract. For use in patients, ionizable lipid nanoparticles rely on the ionizable lipids to be neutrally charged at physiological pH (i.e., 7.4). When the lipid nanoparticles are taken up by cells, they are trapped in endosomes which become increasingly acidic to degrade the endosome components. The ionizable lipids are designed to ionize, that is, become positively charged in the acidic endosome to cause endosome membrane rupture, releasing nucleic acid cargo into the cytoplasm to allow therapeutic effect. On leaving the stomach, the pH gradually increases in the small intestine from 6 to about 7.4 in the terminal ileum. The pH drops to 5.7 in the cecum, but again gradually increases, reaching 6.7 in the rectum. The acidic conditions of the gastrointestinal tract are problematic for lipid nanoparticle function as the acidity will cause premature ionization of the lipid nanoparticles. If not addressed, the lipid nanoparticles would ionize and release nucleic acid cargo in the gastrointestinal tract before entering cells. To overcome the stomach barrier, enteric formulations, such as enterically-coated capsule technology, can be used to package lipid nanoparticles for release of dry or liquid lipid nanoparticle formulations into the intestines. Suitable enteric-release formulations are known and may comprise polymeric compositions or other enteric coatings that are stable at the acidic pH of the stomach, but dissolve in the neutral-to- basic pH of the small intestine. Non-limiting examples of enteric coatings include, without limitation, methyl acrylate-methacrylic acid copolymers, methyl methacrylate- methacrylic acid copolymers, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, shellac cellulose acetate trimellitate, sodium alginate, zein, among others. In one example, a suitable enteric coating is an anionic acrylate polymers, such as EUDRAGIT® compositions (Evonik Industries AG), as are broadly-known and which are tested in the examples below. For example, EUDRAGIT L100-55 is a copolymer based on methacrylic acid and ethyl acrylate used for enteric and delayed release. Different copolymer compositions have different delay and release profiles, and can be used in the LNP formulations described herein singularly or in combination with other enteric polymers. To overcome the acidity barrier of the intestines, inclusion of additives such as bases or buffers to neutralize the intestinal acid may be incorporated into a suitable formulation, drug product, or dosage form. The bases and buffers may be generally recognized as safe (GRAS). Suitable bases may include bicarbonates, carbonates, hydroxides, or others, such as sodium bicarbonate, ammonium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, aluminum hydroxide, magnesium hydroxide, calcium hydroxide, magnesium carbonate, sodium carbonate, potassium carbonate calcium carbonate, trisodium phosphate and/or sodium benzoate. The base or buffer may be provided in any drug product, such as an enteric formulation, in an effective amount, such as from 1 to 99% by weight of the formulation, or from 1 mg to 2 g in a unit dosage form. [0079] By “neutralize” it is meant to raise the pH of an acidic solution, or lower the pH of a basic solution towards pH 7 or towards a physiological pH (e.g., ~7.4), for example and without limitation to raise an acidic pH of less than 6.5 to a pH between 6.5 and 8, and any increment therebetween, for example, ranging from pH 6.8 to 7.6, e.g., 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, or 7.6.

[0080] A delayed-release dosage form (drug product) is provided comprising a lipid- containing particle complexed with an active agent (e.g., a cargo), a base or neutral buffer, and, optionally, an enteric or delayed-release coating or matrix. The lipid- containing particle may be a particle as described herein, e.g., a negatively-charged lipid-containing particle as described herein that is suitable for delivery to mucosa. [0081 ] FIG. 5 is a schematic diagram of a simple oral drug product for enteric or timed- release delivery of a therapeutic agent. The product 10 may be a tablet comprising an enteric or timed-release coating 12 over a core 14. The coating 12 may be any effective coating for release of the core on entry into a patient’s intestines following oral ingestion, such as an enteric coating. The core 14 may comprise a solid matrix, or may comprise a powder or beads, comprising the lipid particles, e.g., lipid nanoparticles, as described herein. The lipid particles may be incorporated into coated particles within the core and may be mixed with a base or buffer, (e.g., an antacid, such as sodium bicarbonate), as described herein. Alternatively, the core 14 may be physically divided into a lipid particle-containing compartment, and an antacid- or buffer-containing compartment. For example, the lipid particles may be contained in a single or multiple particles within the antacid component of the core, and the particles containing the lipid particles dissolve in the intestine after release of the antacid or buffer.

[0082] It is noted that certain bases may also provide active gas propulsion to the formulation. The combination of a carbonate or bicarbonate with an acid reacts to produce carbon dioxide bubbles. Inclusion of a bicarbonate or carbonate in lipid nanoparticle formulations introduced in the gastrointestinal tract can mix with the intestinal acid to both neutralize acid and to produce carbon dioxide gas bubbles that can help actively propel lipid nanoparticles through the mucin.

Example 1

[0083] Lipid nanoparticles were formulated with different helper lipids and used to transfect Caco-2 intestinal epithelial cells covered with a physiological thickness and concentration of mucin with Firefly luciferase mRNA (See, FIG. 6). Using the Bright- Glo™ luminescence assay, transfection was detected by measuring the light signal produced from the Luciferase protein expression. It was observed that lipid nanoparticles formulated with a net negatively charged helper lipid, phosphatidylserine (PS), effectively transited across the mucus barrier and transfected Caco-2 intestinal epithelial cells (FIG. 7). Lipid nanoparticles were then formulated with 40 mol% helper lipid, 35 mol% ionizable lipidoid, 22.5 mol% cholesterol and 2.5 mol% CI4-PEG2000- PE with the lipidoid 3060io, referring to N,N-Bis(3-aminopropyl)methylamine conjugated to decyl acrylate, as described herein.

[0084] Lipid nanoparticles were then formulated with 40 mol% phosphatidylserine, 35 mol% ionizable lipidoid, 22.5 mol% cholesterol and 2.5 mol% CI4-PEG2000-PE with different lipidoids from a library of lipidoids (See FIGS. 2A-4). We identified 11 formulations (500Xi, 5000no, 306On, 3O6O12, 200CQ, 5160no, 500Oi,i,s, 3060io, 514X6, 306OI4, 5OIX1) that effectively transfected Caco-2 cells alone and 8 formulations (3O6O12, 501Xi, 500Xi, 306On, 3060io, 1 130MO, 5160MO, 200CQ) that effectively transfected Caco-2 cells covered with mucin. The top 20 lipidoids in Caco 2 cells without mucin (in order from highest-to-lowest) were: 500Xi; 5000no; 306On; 3O6O12; 200CQ; 5160iio; 500Oi,i,s; 3060io; 514CQ; 306OM; 501 Xi ; 205OIQ; 5000i3; 1130iio; 306OIQ; 3O6O13; 205OI8; 509XZ; 501 Ono; and 5000 . The top 20 lipidoids in Caco2 cells with mucin (in order from highest-to-lowest) were: 3O6O12; 501 Xi ; 500Xi ; 3O6O11; 3O6O10; 1130iio; 5160iio; 200CQ; 5000iio; 509Xi; 509X3; 501 X2; 4020Q,IO; 51604,8; 501 Ono; 402Xs; 501 Oi,i,b; 509Oi,i,b; 5OOO13; and 1130io.

Example 2 - surface ionization of LNP nanoparticles

Materials

[0085] CleanCap® Firefly Luciferase mRNA (L-7602) was purchased from TriLink Biotechnologies. 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (850725P), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), L-a-phosphatidylserine (Brain, Porcine) (sodium salt) (840032P), 1 ,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (PA) (840875P), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (sodium salt) (PG) (840475P), Sphingomyelin (Brain, Porcine) (860062P), N-oleoyl-D-erythro- sphingosine (Cer) (860519P), 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) (890890P) and 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000) (880150P) were purchased from Avanti Polar Lipids. Cholesterol (C8667), sodium citrate monobasic (71497) and Sodium 2-(p-toluidino)-6-naphthalenesulfonic acid (T9792) were purchased from Sigma Aldrich. Quant-iT™ RiboGreen™ RNA Assay Kit (R11490) was purchased from ThermoFisher Scientific.

Lipid Nanoparticle Formulation

[0086] Lipidoids, helper lipids, cholesterol, and C14-PEG2000 were dissolved in reagent grade ethanol at 1 -10 mg/mL. Firefly luciferase mRNA was dissolved in 10 mM sodium citrate monobasic. Lipid solutions were mixed at a 35:16:46.5:2.5 or 35:40:22.5:2.5 lipidoid to helper lipid to cholesterol to PEG molar ratio. Citrate buffer was added to the lipid solutions at 1 :10 volume ratio. The resultant lipid solution was added to an equal volume of RNA solution at a 10:1 lipidoid:mRNA mass ratio and then mixed thoroughly. Finally, an equal volume of phosphate-buffered saline (PBS) was added to the ethanol-citrate mixture, and this was mixed thoroughly. Lipid nanoparticles for in vitro and in vivo studies were formulated at final mRNA concentrations of 5 pg/rrnL and 90 pg/rrnL, respectively. Lipid nanoparticles used for in vivo studies were dialyzed against 2 L of PBS in 3 kDa molecular weight cut off dialysis cassettes for 1 hour. Lipid Nanoparticle Characterization

[0087] Lipid nanoparticles were characterized for size and surface zeta potential using a Malvern ZetaSizer Nano (Malvern Instruments). Prior to analysis by ZetaSizer, the LNPs were diluted ten-fold in PBS to a concentration of 0.5 pg/rnL mRNA. Three technical replicates were conducted on each sample for both size and surface zeta potential. To measure RNA entrapment, intact and lysed nanoparticles were measured for RNA content using a Quant-iT™ RiboGreen™ RNA Assay Kit according to manufacturer instructions. Briefly, LNPs were diluted in equal volumes of Tris-EDTA (TE) buffer or 2% Triton X-100 in TE buffer. Then, an equal volume of RiboGreen™ reagent was added to each sample and incubated at 37 °C for 15 minutes. The fluorescence (ex/em 480/520 nm) was read on a Tecan Spark® Multimode Microplate Reader. To measure Sodium 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) ionization a surrogate measure of LNP surface charge, 5 mI_ nanoparticles were diluted in 250 mI_ TNS assay buffer (20 mM sodium phosphate tribasic, 25 mM ammonium citrate dibasic, 20 mM ammonium acetate, 150 mM sodium chloride) at pH 7.4 or pH 5. Then, 5 mI_ of a 0.16 mM stock solution of 2-(p-toluidinyl)naphthalene-6-sulphonic acid (TNS, Sigma Aldrich) in Dl water was added to each well. The TNS fluorescence was read on a Tecan Spark® (ex/em 320/430 nm).

Cell Culture

[0088] Caco-2 cells were cultured in DMEM/10% FBS/1% Pen/Strep/0.1% Fungizone at 37°C/5% CO2 and split with trypsin prior to confluency. Cells were allowed to adhere or settle for 24 hours prior to LNP administration. A physiologically relevant mucin layer was created by adding 25 pL of 5% mucin diluted in media covered by an additional 155 pL of media. Twenty microliters of LNPs per well at an original concentration of 5 pg/mL Firefly luciferase mRNA (as described above) were added into cell culture media then allowed to incubate for 24 hours. Transfection efficiency was measured using Bright-Glo™ Luciferase Assay System (Promega). Briefly, BrightGlo reagent was diluted 1 :4 in PBS then 50 pL was added to each well. After a 7-minute incubation in the dark, luminescence intensity was read on a Tecan Spark plate reader.

In Vivo Intravenous LNP Administration

[0089] Female C57BL/6NCrl (Charles River) mice of at least 6 weeks of age were used for all in vivo experiments. Mice were anesthetized using isoflurane and maintained under anesthesia for the entirety of the procedure. The abdominal skin was disinfected with isopropanol and then incised to reveal the abdominal muscle. The abdominal muscle was also dissected to reveal the peritoneal organs. The intestines were exposed and the region just distal to the stomach was isolated. The intestines were injected with 50 pL of propulsion agent (10% sodium bicarbonate) immediately prior to LNP administration and just distal of the stomach. Mice then received intestinal injections of Firefly luciferase imRNA-containing LNPs at a dose of 0.75 mg/kg at the same site of propulsion injection. Three hours later, mice were injected intraperitoneally with 130 mI_ of 30 mg/mL D-luciferin. Fifteen minutes following luciferin administration, mice were euthanized via CO2 asphyxiation and secondary cervical dislocation. Organs were removed, excess blood was blotted off, and organs were placed on black construction paper. Luminescent signal was measured using an In Vivo Imaging System (Perkin Elmer), and luminescent images were juxtaposed with brightfield images. Total luminescent flux (p/s) was calculated for each organ using Living Image® software. Organs used for histology were immediately placed in 10% neutral buffered formalin and stored at 4 °C for 4 days before washing in PBS and storing in 70% ethanol at 4 °C. Organs used for flow cytometric analysis were placed into cold DMEM during transport to the cell culture room for processing.

[0090] In FIG. 8A, we plotted the in vitro efficacy of LNPs used to treat Caco-2 intestinal epithelial cells versus the ionization of the lipid nanoparticles as assessed by TNS fluorescence. The most efficacious LNPs all had lower ionization values on the range observed, suggesting that increased negative charge was beneficial for Caco- 2 intestinal epithelial cell efficacy. Furthermore, all LNPs with higher ionization values had poor transfection, suggesting that more positive charge was detrimental to Caco- 2 transfection.

[0091] In FIG. 8B, we plotted Caco-2 intestinal cell efficacy versus ionization of LNPs at pH 5 (the pH of endosomes that LNPs must escape to allow mRNA translation). While there were similar trends to pH 7, they were also weaker than at pH 7, suggesting that the charge at pH 7 is more important for transfection of intestinal epithelial cells in vitro.

[0092] In FIG. 9A, we plotted the efficacy of LNPs in transfecting Caco-2 intestinal epithelial cells covered with a layer of mucin versus the ionization at pH7 as assessed by TNS fluorescence. The trends from the experiment involving uncovered Caco-2 cells continued with greater effect. Only LNPs in the lower third of observed TNS fluorescence values had increased efficacy compared to the rest of the population. This suggests that negative charge is increasingly important for LNPs to cross a mucin layer and transfect intestinal epithelial cells.

[0093] In FIG. 9B, we plotted the efficacy of LNPs transfected Caco-2 cells covered in a layer of mucin versus the LNP ionization at pH 5. Similar to pH 7, the most efficacious LNPs had lower ionization values. This trend was less pronounced than at pH 7, again suggesting that the ionization at pH 7 is more important than at pH 5 for mucus transit and intestinal epithelial cell transfection.

[0094] In FIG. 10, we assessed the ionization of LNPs formulated with different helper lipids at low concentrations (16 mol%) and high concentrations (40 mol%) of helper lipids. DOPE, DOPC, SM, and Cer are neutrally charged helper lipids. PS, PA, and PG are negatively charged helper lipids. DOTAP and EPC are positively charged helper lipids. As can be seen in the 16 mol% helper lipid graphs, only DOTAP had a significant effect on LNP ionization. At 40% helper lipid, however, all the negatively charged helper lipids lowered ionization values compared to neutrally charged helper lipids.

Example 3

[0095] Different combinations of acids and carbonates were tested to create a gas propulsion chemistry to actively propel lipid nanoparticles across the mucus barrier, essentially as described above. Ten milligrams of propulsion powder was added to each well and incubated for four hours. The acids and carbonates are described in connection with FIG. 11. Transwells were coated with a physiologically relevant concentration and thickness of mucin. Lipid nanoparticles that encapsulated a fluorescent mRNA cargo were added in the top compartment of transwells. Then gas propulsion chemistries were added in the top compartment as well. The bottom compartment was assayed for fluorescence every hour for four hours. As depicted in FIG. 11, we discovered formulations that enhanced lipid nanoparticle transit across the mucus barrier up to 11 times that observed without propulsion. Combinations of sodium bicarbonate and sodium citrate (NaaCitrate) were found to have the highest efficacy of propelling the fluorescently labeled nanoparticles across the mucin-coated transwells.

[0096] Lipid nanoparticles were tested in vivo by direct intestinal injection. Lipid nanoparticles were formulated with 40 mol% phosphatidylserine, 35 mol% 3060io, 22.5 mol% cholesterol and 2.5 mol% C14-PEG2000 . Mice were anesthetized with isoflurane then their abdomen was incised open to expose the intestines. A sodium bicarbonate solution (amount, concentration) was injected into the intestine below the stomach to neutralize the intestinal acid. Lipid nanoparticles were then injected into the intestines in the same location. The abdomens were sealed with tissue adhesive and the mice were maintained under anesthesia for four hours. The mice were then injected intraperitoneally with D-luciferin for 15 minutes and then euthanized. The intestines were harvested and imaged using an In Vivo Imaging System (IVIS). The imaging revealed transfection throughout the entire length of the intestines (See,

FIG. 12).

[0097] Imaging of the major organs from these mice revealed that the liver, spleen and pancreas were also transfected following intestinal injection, suggesting the lipid nanoparticles crossed the intestine and entered the bloodstream, allowing transfection similar to that observed upon intravenous administration of lipid nanoparticles

(FIG. 13).

[0098] Quantification of experimental results showed that there is consistent, repeatable transfection of the intestine, liver and pancreas following intestinal injection. FIGS. 14-17 show quantification of transfection efficiency in the intestine, liver, spleen, and pancreas, respectively. Photon flux (p/s) was measured using an In Vivo Imaging System set to capture luminescence intensity (excitation off, emission open). The exposure was set to auto.

Example 4 - evaluation of helper lipids and enteric polymers [0099] LNPs were formulated with different helper lipids (DOPE, DOPS, DOPG, DOPA, and DOTAP) at 35 mol% lipidoid (3060io), 40 mol% helper lipid, 22.5% cholesterol, 2.5% C14-PEG2000 with a lipidoid to mRNA ratio of 10 and mRNA cargo of mFLuc. Polymers were added to the LNP solutions to make a final polymer concentration of 1% (w/v). Polymers included common enteric excipients: carboxymethylcellulose, EUDRAGIT L100, EUDRAGIT L100-55, and EUDRAGIT S100. Caco-2 cells (100,000 per well) were seeded in white 96 well plates and cultured for 24 hours then LNPs were added and incubated for 24 hours. BrightGlo luminescence assay was performed and read on a spectrophotometer according to manufacturer instructions. Results are shown in FIG. 18.

[00100] LNPs were formulated with different helper lipids (DOPE, DOPS, DOPG, DOPA, and DOTAP) at 35 mol% lipidoid (3060io), 40 mol% helper lipid, 22.5% cholesterol, 2.5% C14-PEG2000 with a lipidoid to mRNA ratio of 10 and mRNA cargo of mFLuc. Polymers were added to the LNP solutions to make a final polymer concentration of 1% (w/v). Polymers included: carboxymethylcellulose, EUDRAGIT L100, EUDRAGIT L100-55, and EUDRAGIT S100. Caco-2 cells (100,000 per well) were seeded in white 96 well plates and cultured for 24 hours. Cells were covered with 25 pL of 5% mucin in media then 155 pL normal media. LNPs were added and incubated for 24 hours. BrightGlo luminescence assay was performed and read on a spectrophotometer according to manufacturer instructions. Results are shown in

FIG. 19.

Example 5 - evaluation of buffers/antacids

[00101] LNPs were formulated with 35 mol% lipidoid (3060io), 40 mol% DOPS, 22.5% cholesterol, 2.5% C14-PEG2000 with a lipidoid to mRNA ratio of 10 and mRNA cargo of mFLuc. LNPs were formulated in different buffers/antacids, as indicated, to observe their effect on transfection. Polymers were added to the LNP solutions to make a final polymer concentration of 1% (w/v). Polymers included: carboxymethylcellulose, EUDRAGIT L100, EUDRAGIT L100-55, and EUDRAGIT S100. Caco-2 cells (100,000 per well) were seeded in white 96 well plates and cultured for 24 hours then LNPs were added and incubated for 24 hours. BrightGlo luminescence assay was performed and read on a spectrophotometer according to manufacturer instructions. Results are shown in FIG. 20.

[00102] LNPs were formulated with 35 mol% lipidoid (3060io), 40 mol% DOPS, 22.5% cholesterol, 2.5% C14-PEG2000 with a lipidoid to mRNA ratio of 10 and mRNA cargo of mFLuc. LNPs were formulated in different buffers/antacids to observe their effect on transfection. Polymers were added to the LNP solutions to make a final polymer concentration of 1% (w/v). Polymers included: carboxymethylcellulose, EUDRAGIT L100, EUDRAGIT L100-55, and EUDRAGIT S100. Caco-2 cells (100,000 per well) were seeded in white 96 well plates and cultured for 24 hours. Cells were covered with 25 pL of 5% mucin in media then 155 pL normal media. LNPs were added and incubated for 24 hours. BrightGlo luminescence assay was performed and read on a spectrophotometer according to manufacturer instructions. Results are shown in FIG. 21.

Example 6 - substitution of cholesterol with fatty acids and bile acids [00103] LNPs were formulated essentially as described in Examples 4 and 5, but with partial replacement of cholesterol with a fatty acid or bile acids. _35% lipidoid / 16% helper lipid / 46.5% cholesterol (or bile acid or fatty acid/cholesterol mixture) / 2.5% C14-PEG2000. Partial replacement of cholesterol with fatty acids maintains transfection of Caco-2 cells (FIG. 22) and Caco-2 cells covered with mucin (FIG. 23). Different amounts of phosphatidyl serine (PS) and phosphatidyl ethanolamine (PE) were used in the fatty acid formulations. DOPE and phosphatidyl serine were added as helper lipids for the bile acid substitution. For the bile acid substitutions, cholesterol was replaced completely with the indicated bile acids. Replacement of cholesterol with bile acids maintains transfection in Caco-2 cells (FIG. 24). Replacement of cholesterol with bile acids potentially improves formulations with PS but does not significantly improve transfection of formulations with DOPE in Caco-2 cells covered with mucin

(FIG. 25).

Example 7 - LNP oral gavage

Lipid nanoparticles were formulated with 35 mol% 3060no / 40 mol% DOPS / 22.5 mol% cholesterol / 2.5 mol% C14-PEG2000 with Firefly Luciferase mRNA at a lipidoid:mRNA ratio of 10 (w/w). Lipid nanoparticle solutions were combined with enteric polymers such as carboxymethyl cellulose or Eudragit L100, L100-55 or S100 to achieve a final mass concentration of 1%. Lipid nanoparticle solutions were then dialyzed with PBS or antacid solutions such as sodium bicarbonate or calcium carbonate to achieve antacid concentrations from 1 -10%.

[00104] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.

Claims

Claims:

1 . A trans-mucosal drug delivery method for delivery of a therapeutic agent to a patient, comprising administering to mucosa of a patient a composition comprising a lipid-containing particle, such as a lipid nanoparticle, comprising a therapeutic agent, the lipid-containing particle comprising: a helper lipid having a negative charge at pH7; cholesterol or a derivative thereof; a PEG-based compound, such as a PEG-containing polymer or a PEGylated fatty acid-containing compound; and an ionizable lipidoid, e.g. that forms a cation at an acidic pH; wherein the lipid-containing particle has a negative surface charge at pH 7 and, optionally, at pH 5.

2. The method of claim 1 , wherein the negative surface charge is determined by a 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) fluorescence assay.

3. The method of claim 2, wherein the pKa of the particle is less than 5 as determined by TNS fluorescence.

4. The method of claim 1 , wherein the ionizable lipidoid is 306OM 0.

5. The method of any one of claims 1 -4, wherein the helper lipid is an amphipathic glyceride, phospholipid, or phosphoglyceride.

6. The method of claim 5, wherein the helper lipid is a phosphatidyl serine.

7. The method of any one of claims 1 -6, wherein the therapeutic agent is anionic or polyanionic.

8. The method of claim 7, wherein the therapeutic agent is a nucleic acid or protein.

9. The method of claim 8, wherein the nucleic acid is an RNA.

10. The method of claim 9, wherein the RNA is an mRNA.

11 . The method of claim 9, wherein the RNA is an RNA reagent chosen from an RNAi reagent, a dsRNA, an siRNA, an shRNA, a miRNA, an antisense RNA, a guide RNA (gRNA), a long non-coding RNAs (IncRNA), a base editing gRNA (beRNA), a prime editing gRNA (pegRNA), or a transfer RNA (tRNA).

12. The method of any one of claims 1 -11 , wherein the PEGylated fatty acid-containing compound compound is a PEGylated C10-C20 fatty acid- containing compound, such as C14-PEG2000-PE.

13. The method of any one of claims 1 -12, wherein the lipid- containing particle is a lipid nanoparticle comprising: from 10% mol. to 80% mol. of the helper lipid; from 5% mol. to 50% mol. of the cholesterol; from 0.5% mol. to 20% mol. of the PEGylated fatty acid-containing compound ; and from 20% mol. to 60% mol. of the ionizable lipidoid.

14. The method of any one of claims 1 -13, wherein the helper lipid is phosphatidyl serine; the PEGylated fatty acid-containing compound is a C10-C16 fatty acid-containing compound, such as C14-PEG2000-PE.

15. The method of any one of claims 1 -14, wherein the ionizable lipidoid is one or more of 500Xi; 5000no; 306On; 306OM 0; 3O6O12; 200CQ; 5160no; 5000-1,1,8; 3060io; 514X6; 306O ; 501 Xi ; 205OIQ; 5000I3; 1130no; 306OIQ; 306OI3; 205OIS; 509Xz; 501 Oiio; 5030no; 5000 ; 1 130MO; 509XI ; 509X₃; 501 X2; 4020e,io; 51604,8; 402X8; 501 Oi,i,s; or 509Oi,i,s.

16. The method of any one of claims 1 -14, wherein the ionizable lipidoid is one or more of 500Xi, 5000no, 306On, 3O6O12, 200CQ, 5160MO, 5000I,I,S, 3060io, 514Xe, 306014, 5OIX1, 1130no, or 200X₆.

17. The method of any one of claims 1 -14, wherein the ionizable lipidoid is the reaction product of an amine and an acrylate, wherein the amine is 306, 205, 200, or 304, and the acrylate has the formula:

where n ranges from 8 to 13, and is saturated or unsaturated, e.g., having from one to three double bonds.

18. The method of any one of claims 1 -7, wherein the mucosa is intestinal mucosa.

19. The method of claim 18, wherein the lipid-containing particle comprising the therapeutic agent is administered in an oral enteric or timed-release unit dosage form.

20. The method of claim 18 or 19, further comprising administering a buffer or base with the lipid-containing particle comprising the therapeutic agent, to neutralize acid in the patient’s intestine.

21 . The method of claim 20, wherein the buffer or base is contained in an oral enteric or timed-release unit dosage form with the lipid-containing particle comprising the therapeutic agent, wherein optionally the base or buffer is from 1 to 99% by weight of the formulation, or from 1 mg to 2 g.

22. The method of claim 20 or 21 , wherein the buffer or base is a bicarbonate, such as sodium bicarbonate, magnesium bicarbonate, or aluminum bicarbonate.

23. The method of claim 22, wherein the buffer or base is NaHC03 contained in an oral enteric or timed-release unit dosage form with the lipid-containing particle comprising the therapeutic agent and an acrylic polymer, such as EUDRAGIT L100-55, and wherein the ionizable lipidoid is 306OM0.

24. An oral drug product, comprising: a core comprising a lipid-containing particle, such as a lipid nanoparticle, comprising a therapeutic agent, the lipid-containing particle comprising: a helper lipid having a negative charge at pH7; cholesterol; a PEGylated fatty acid-containing compound , such as a PEG-containing polymer, or a PEGylated phospholipid; and an ionizable lipidoid, e.g., that forms a cation at an acidic pH; wherein the lipid-containing particle has a negative surface charge at pH7 and pH 5; and an enteric or timed-release coating covering the core.

25. The drug product of claim 24, further comprising a base or buffer, optionally in an amount effective to neutralize intestinal acid, such as from 1 to 99% by weight of the formulation, or from 1 mg to 2 g.

26. The drug product of claim 25, wherein the buffer or base is sodium carbonate, calcium carbonate, or a bicarbonate, such as sodium bicarbonate, magnesium bicarbonate, or aluminum bicarbonate.

27. The drug product of claim 25, wherein the buffer or base is NaHC03 contained in an oral enteric or timed-release unit dosage form with the lipid- containing particle comprising the therapeutic agent, and an acrylic polymer, such as EUDRAGIT L100-55, and wherein the ionizable lipidoid is 3060iio.

28. The drug product of any one of claims 24-27, wherein the negative surface charge is determined by a 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) fluorescence assay.

29. The drug product of any one of claims 24-28, wherein the pKa of the particle is less than 5 as determined by TNS fluorescence.

30. The drug product of any one of claims 24-29, wherein the helper lipid is an amphipathic glyceride, phospholipid, or phosphoglyceride.

31. The drug product of claim 30, wherein the helper lipid is a phosphatidyl serine or a phosphatidyl ethanolamine.

32. The drug product of any one of claims 24-31 , wherein the therapeutic agent is anionic or polyanionic.

33. The drug product of claim 32, wherein the therapeutic agent is a nucleic acid.

34. The drug product of claim 33, wherein the nucleic acid is an RNA.

35. The drug product of claim 34, wherein the RNA is an mRNA.

36. The drug product of claim 34, wherein the RNA is an RNA reagent chosen from an RNAi reagent, a dsRNA, an siRNA, an shRNA, an miRNA, an antisense RNA, a guide RNA (gRNA), a long non-coding RNAs (IncRNA), a base editing gRNA (beRNA), a prime editing gRNA (pegRNA), or a transfer RNA (tRNA).

37. The drug product of any one of claims 24-36, wherein the PEG- based compound is a PEGylated C10-C16 fatty acid-containing compound , such as a PEGylated phospholipid, e.g., a PEGylated C -phosphatidylethanolamine.

38. The drug product of any one of claims 24-37, wherein the lipid- containing particle is a lipid nanoparticle comprising: from 10% mol. to 80% mol. of the helper lipid; from 5% mol. to 50% mol. of the cholesterol; from 0.5% mol. to 20% mol. of the PEGylated fatty acid-containing compound ; and from 20% mol. to 60% mol. of the ionizable lipidoid.

39. The drug product of any one of claims 24-38, wherein the helper lipid is phosphatidyl serine; the PEGylated fatty acid-containing compound comprises a C10-C16 fatty acid, such as C14-PEG2000.

40. The drug product of any one of claims 24-39, wherein the ionizable lipidoid is one or more of 500Xi; 5000no; 306On; 3060iio; 3O6O12; 200CQ; 5160iio; 5000-1,1,8; 3060io; 514CQ; 306OM; 501 Xi ; 205OIQ; 5000I3; 1130ho; 306OIQ; 306OI₃; 205OIS; 509XZ; 501 OMO; 5030MO; 5000M; 1 130MO; 509XI ; 509X₃; 501 X2; 4020Q,IO; 51604,8; 402Xs; 50101 ,1 ,s; or 509Oi,i,s.

41. The drug product of any one of claims 24-39, wherein the ionizable lipidoid is one or more of 500Xi, 5000no, 306On, 3O6O12, 200CQ, 5160MO, 5000I,I,8, 3060IO, 514X6, 306014, 501Xi, 1130ho, or 200Xe.

42. The drug product of any one of claims 24-39, wherein the ionizable lipidoid is the reaction product of an amine and an acrylate, wherein the amine is 306, 205, 200, or 304, and the acrylate has the formula:

where n ranges from 8 to 13, and is saturated or unsaturated, e.g., having from one to three double bonds.

43. The drug product of any one of claims 24-42, wherein the buffer or base is NaHCC>3 contained in an oral enteric or timed-release unit dosage form with the lipid-containing particle comprising the therapeutic agent, and an acrylic polymer, such as EUDRAGIT L100-55, and wherein the ionizable lipidoid is 3060iio.

44. The drug product of any one of claims 24-43, wherein the mucosa is intestinal mucosa.